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Bi 1 Lecture 3

Thursday, March 30, 2006

What is a Receptor?

Receptors and Ion Channels as Examples of Proteins

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receptor

Most drug receptors are proteins.

a molecule on the cell surface or in the cell interior that has an affinity for a specific molecule (the ligand).

Latin, “to tie”

Greek, “first”

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side chains

“peptide”or

amide bonds

link the

“backbone”or

“main chain”or

“-carbons”

Little Alberts Figure 2-22© Garland publishing

shortest: 9longest: 5500

20 types

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helices sheets

http://www.its.caltech.edu/~lester/Bi-1/alpha-helix-alphabetical.pdb

http://www.its.caltech.edu/~lester/Bi-1/beta-sheet-antiparallel.pdb

Proteins contain a few structural motifs:

Hide side chainsShow H-bonds and distancesShow ribbons & arrowsShow side chainsShow Van der Waals radii

(Swiss-prot viewer must be installed on your computer)

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nicotinic acetylcholine

receptor

Most drug receptors are membrane proteins

Outside the cell

Inside the cell = cytosol

(view in ~1995)

natural ligand(agonist)

nicotine, another agonist

Membrane = lipid bilayer

~ 100 Å= 10 nm

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Overall topology of the nicotinic acetylcholine receptor(view in ~2000)

outside the cell:

5 subunitseach subunit has 4 -helices

in the membrane (20 membrane helices total)

Little Alberts figure 12-42© Garland publishing

Binding Region

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The acetylcholine binding protein (AChBP) from a snail, discovered in 2001, strongly resembles the binding region

(Swiss-prot viewer must be installed on your computer)

Color by chainShow 2 subunits,Chains,Ribbons

5 subunits

Little Alberts figure 12-42© Garland publishing

http://www.its.caltech.edu/~lester/Bi-1/AChBP+Carb-5mer.pdb

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http://www.its.caltech.edu/~lester/Bi-1-2004/AChBP-2004-BindingSite.pdb

The AChBP binding site occupied by an acetylcholine analog (2004)

http://www.its.caltech.edu/~lester/Bi-1/AChBP-2004-BindingSite.pdb

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Binding region

Membrane region

Cytosolicregion

Colored by secondary

structure

Colored by subunit(chain)

Nearly Complete Nicotinic Acetylcholine Receptor (February, 2005)

http://pdbbeta.rcsb.org/pdb/downloadFile.do?fileFormat=PDB&compression=NO&structureId=2BG9

~ 2200 amino acids in 5 chains

(“subunits”),

MW ~ 2.5 x 106

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How the binding of agonist (acetylcholine or nicotine) might open the channel: June 2003 view

M2

M1

M3

M4

Ligand-bindingregion

11Nicotinic acetylcholine receptor

Most drug receptors are membrane proteins

Some drugs bind on the axis

Some drugs compete with nicotine or acetylcholine

membraneregion

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Protein Lecture #

Ligand-gated Ion Channels 3 (today)

Pumps and transporters 5, 13

Motors 10

G protein-coupled receptors and G proteins 12

Enzymes 13, 15

DNA-binding proteins 18

RNA polymerase, ribosome 18

Cystic Fibrosis Transmembrane Regulator 20

Rhodopsin 26

All I really need to know about lifeI learned in Bi 1

1. If you want a job done right, get a protein

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Protein structure prediction:

An important 21st-century problem

Want to test your own skill at predicting protein structure?

Then enter “Critical Assessment of Techniques for Structure Prediction”or CASP 7

http://predictioncenter.org/

Winners earn an automatic “A+” in Bi1 (retroactively, if appropriate)

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Protein Folding vs. “Inverse Folding” = Computational Protein Design

Protein Folding(no degeneracy)

Inverse Folding(large degeneracy)Set of All

StructuresSet of All

Sequences

Individualamino acids

Several ways to make an arch

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X-ray Crystallography

Crystal Growth

X-ray Data

Electron Density

Protein Model

http://www.search.caltech.edu/CIT_People/action.lasso?-database=CIT_People&-response=Detail_Person.html&-layout=all_fields&person_id=29067&-search

Bi 1 Cameo by Professor Pamela J. Bjorkman

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X-ray crystallography

• Why X-rays?Right wavelength to resolve atoms

• Why crystals?Immobilize protein, enhance weak signal from scattering

• What is a protein crystal?Large solvent pathways (20-80% solvent)Same density as cytoplasmEnzymes active in crystals

• Are crystal structures valid compared with solution structures?Usually -- Compare NMR and X-ray structuresStructures correlate with biological functionMultiple crystal forms look same -- small effects of

packing

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Overview of imaging

No lens to refocus X-rays, so must

understand reciprocal space and

diffraction

Diffraction:Scattering followed

by interference

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Bragg’s lawConsider simultaneous reflection of a large number of x-rays.

See diffraction maximum in direction only if diffracted waves are in phase.

Path difference (2dsin) must represent an integral number of wavelengths to get

constructive interference.

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Learned two things from Bragg’s Law

• sin = n/2 x 1/d

Low angle: large interplanar spacing

High angle: small interplanar spacing

Since sin 1/d, structures with large interplanar

spacings (d) will have diffraction patterns with small

spacings and vice-versa.

• Repeating unit in real space (crystal) --> diffraction

maxima and minima

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Same molecular transform sampled by different lattices

Modified from Lipson & Taylor, 1964

a) Molecular transform b) Lattice

d - f ) Same molecular transform sampled by different lattices

c) Convolution of lattice and transform

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Resolution

From Harburn, Taylor, Wellbery, An Atlas of Optical Transforms

An inverse Fourier transform (FT) including all of the high angle information gives back the original image.

An inverse FT including only the low angle information gives back a low resolution view of Mickey.

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The electron density equation and the phase problem

• There are experimental methods for

determining the phase for each reflection hkl.

(xyz ) 1

V h

k | F(hkl) | exp[( 2i(hk ky lz ) ihkl]

l

Can measure this|F(hkl)|=I1/2

Can’t measure this

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X-ray detection

• Film (relic of the past)

• Diffractometers (almost relic

of past, but used for small

molecules)

• Multiwire detectors (almost

relic of past)

• Phosphorimager detectors

(R-AXIS, MAR)

• CCD detectors

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Synchrotron x-ray sources

• High-intensity x-ray emitted by charged particles accelerated in a curved path

• X-ray wavelength in range of 0.5 - 2 Å (from E=h=hc/)

• Is tunable!!

Radiation emitted by accelerating charged particle

tangent to path of circle

e-